Regulatory nucleic acid sequences and uses in actinomycetes

ABSTRACT

PCT No. PCT/FR94/01413 Sec. 371 Date Jun. 7, 1996 Sec. 102(e) Date Jun. 7, 1996 PCT Filed Dec. 5, 1994 PCT Pub. No. WO95/16046 PCT Pub. Date Jun. 15, 1995Novel nucleic acid sequences, vectors for expressing same, and uses of said sequences, in particular in actinomycetes fermentation methods.

The present invention relates to a new nucleic acid sequence, to vectorsfor its expression and to its use in fermentation processes inactinomycetes.

Actinomycetes are branched filamentous Gram-(+) bacteria. Amongactinomycetes, streptomycetes represent the largest family.Streptomycetes are spore-forming filamentous bacteria which livenaturally in the soil under strictly aerobic conditions.

Actinomycetes, and streptomycetes in particular, are of great importancefrom an industrial standpoint. In particular, they possess the featureof producing a wide variety of secondary metabolites (Demain, Biology ofActinomycetes 88, Okami (Eds), Tokyo, Japan scientific societies press,1988, p. 19-25). These metabolites can have very different structuresand biological properties (herbicides, anticancer agents, anthelmintics,anabolic agents, antibiotics, and the like). The best known of thesemetabolites are antibiotics (chemical substances produced by an organismand having a deleterious effect on other organisms). Streptomyces arecurrently the source of 70% of industrially produced antibiotics. Thestructural diversity of the antibiotics synthesized is not to be foundin any other bacterial genus. Thus, almost all types of structure arerepresented: β-lactams (e.g. ampicillin), polypeptide antibiotics (e.g.streptogramins), aminoglycoside antibiotics (e.g streptomycin,kanamycin, and the like), macrolides (e.g. erythromycin, spiramycin, andthe like) or alternatively cyclines (e.g. tetracycline), and the like.

For these reason, it is especially advantageous to be able to have tools(vectors, promoters, and the like) at one's disposal enabling thesemicroorganisms to be manipulated genetically. Such tools would make itpossible, in effect, to modify the levels of synthesis of thesemetabolites, or to prepare synthesis intermediates or derivatives ofthese metabolites, and the like. Such tools would also make it possibleto make these microorganisms manufacture recombinant products, inparticular heterologous proteins, according to genetic engineeringtechniques.

In this connection, Patent Application No. EP 350,341 describes vectorsderived from plasmid pSAM2 having very advantageous properties. Thus,these vectors are capable of integrating in a site-specific manner inthe genome of actinomycetes, and possess a broad host range and highstability. Moreover, they may be used for transferring nucleic acidsinto actinomycetes and expressing these nucleic acids therein. However,these vectors possess some drawbacks, which lie, in particular, in theirlow copy number per cell, and in the absence of means of controlling thecopy number. Thus, pSAM2 and its derivatives generally integrate on thebasis of a single copy per chromosome.

The present invention supplies a solution to this problem, by providingtools capable of improving the conditions of industrial use of thevectors derived from pSAM2. The present invention describes, in effect,a gene whose expression product leads to the appearance, from integratedforms, of replicative free forms of plasmid pSAM2 or of vectors derivedfrom the latter. This has the effect of increasing the copy number ofpSAM2 or its derivatives, since the free forms are present in a highcopy number per cell.

The present invention also describes cassettes for the expression ofthis gene, vectors containing it and their use for inducing theappearance of free copies of pSAM2 or integrative vectors derived fromthe latter.

The Applicant has, in effect, isolated, sequenced and characterized aregion of plasmid pSAM2 capable of inducing the appearance ofreplicative free copies of pSAM2 or its derivatives. The Applicant hasalso shown that this region could be used in cis (on the same vector) orin trans (not on the same vector) to act on pSAM2 or its derivatives.The sequence of this region is presented in the sequence SEQ ID NOS:1and 2. More specifically, this region and its functional role weredemonstrated by studying variants of plasmid pSAM2: on the one handpSAM2(B2) originating from S. ambofaciens ATCC 15154 for which no freeform is observed, and on the other hand pSAM2 (B3) and pSAM2 (B4)originating from other S. ambofaciens strains for which the free form isobserved with the integrated form. This study enabled two differentpoint mutations to be characterized (those of pSAM2 (B3) and that ofpSAM2 (B4), both localized in the promoter region controlling theexpression of a pSAM2 gene (see SEQ ID NOS:1 and 2. These mutations leadto the appearance of the free form of pSAM2 (B3) and of pSAM2 (B4). Thisgene was then cloned and sequenced, and its capacity to cause theappearance of the free form of plasmids derived from pSAM2 from theintegrated copy was demonstrated.

A first subject of the invention hence lies in a nucleic acid sequencecomprising all or part of the sequence SEQ ID NOS:1 and 2 or of avariant of the latter.

For the purposes of the present invention, the term variant denotes anysequence differing from the sequence SEQ ID NOS:1 and 2 as a result ofthe degeneracy of the genetic code, as well as any sequence whichhybridizes with these sequences or fragments of the latter and whoseproduct possesses the stated activity. These variants may be obtainedfrom SEQ ID No. 1 by any technique known to a person skilled in the art,in particular mutation, deletion, substitution, addition, hybridizationand the like. Hybridizations may be carried out under conventionalconditions of stringency (Maniatis et al., see General techniques ofmolecular biology) or, preferably, under conditions of high stringency.The capacity of the variants to induce the appearance of replicativefree forms of pSAM2 or its derivatives may be determined on anactinomycete strain containing such a plasmid in integrated form (forexample on the strain ATCC 15154), by transfecting the said variant intothe strain under conditions permitting its expression, and verifying theappearance of the free forms (see examples).

Another subject of the invention relates to any cassette for theexpression of the sequence SEQ ID No. 1 or of a variant of the latter asdefined above, comprising the said sequence or variant under the controlof a constitutive or regulated promoter.

The use of a constitutive promoter is especially advantageous when thecassette is used in tans to induce free forms of an integrated plasmid.In this case, the cells containing the integrated form of the plasmidare transfected with the expression cassette to induce the appearance ofreplicative free forms, making it possible, for example, to isolateand/or purify the plasmid.

The use of a regulated promoter is especially advantageous when thecassette is used in cis (on the vector derived from pSAM2 itself), forexample in an industrial fermentation process. In this case, the wholeof the proliferation and growth phase of the cell is performed withoutexpression of the gene of the invention, that is to say with a singlecopy of the plasmid per chromosome, and, for the production phase (of arecombinant product, of a gene for the biosynthesis or regulation of thesynthesis of a metabolite, and the like), the regulated promoter isinduced, bringing about the appearance of several free copies of theplasmid and thus an enhanced production activity. This mode ofimplementation is especially advantageous when the vector derived frompSAM2 carries heterologous sequences whose presence in several number ofcopies may be toxic to the cells and/or affect their growth.

Among constitutive promoters which can be used in the context of thepresent invention, mention may be made more especially of anyconstitutive promoter which is functional in actinomycetes, such as, forexample, the promoter of the ermE gene or a variant of the latter (Bibbet al., Gene 41 (1986) E357), the p14 promoter of phage I19 of S.ghanaensis (Labes et al., Sixth DFGWT/AFAST, 27-30/11/92), or anyfragment containing a promoter region of a ribosomal operon of S.ambofaciens (Pernodet et al., Gene 79 (1989) 33).

Among regulable promoters which can be used in the context of thepresent invention, mention may be made more especially of any regulablepromoter which is functional in actinomycetes. These can comprisepromoters induced specifically by an agent introduced into to theculture medium, such as, for example, the thiostrepton-induciblepromoter tipA (Murakami et al., J. Bactt., 171 (1989) 1459), orthermoinducible promoters such as that of the groEL genes, for example(Mazodier et al., J. Bact. 173 (1991) 7382). They can also comprise anactinomycetes promoter which is specifically active in the late phasesof the proliferation cycle of actinomycetes, such as, for example,certain promoters of genes of the secondary metabolism (genes for theproduction of antibiotics, in particular).

Another subject of the invention relates to the use of the sequences ofthe invention as defined above, or of cassettes containing them, forinducing the appearance of free copies of vectors which are derived frompSAM2 and integrated in an actinomycete.

As stated above, this use may be performed in cis (the gene or cassettebeing carried by the integrative vector derived from pSAM2) or in trans(the gene or cassette being on another vector or even introduceddirectly as such).

The integrative vectors derived from pSAM2 as mentioned above arevectors comprising at least the elements of pSAM2 needed forintegration, excision and replication. More especially, these vectorshence comprise at least the attP and int regions as described inApplication EP 350,341, the Xis gene, the repSA gene and the origin ofreplication (ori). These different regions are shown on the map ofplasmid pSAM2 given in FIG. 1, on which some restriction sites enablingthese regions to be extracted also appear.

Advantageously, the integrative vectors derived from pSAM2 also comprisea recombinant DNA sequence coding for a desired product. The latter canbe a peptide, polypeptide or protein of pharmaceutical oragri-foodstuffs importance. In this case, the system of the inventionmakes it possible to increase the copy number of this sequence per cell,and hence to increase the levels of production of this product and thusto increase the yields of the preparation process. The desired productcan also be a peptide, polypeptide or protein participating in thebiosynthesis (synthesis, degradation, transport or regulation) of ametabolite by the actinomycete strain in question. In this case, thesystem of the invention makes it possible to increase the copy number ofthis sequence per cell, and hence to increase the levels of productionof this product, and thus either to increase the levels of production ofthe metabolite, or to block the biosynthesis of the metabolite, or toproduce derivatives of the metabolite.

The sequences of the invention may thus be used in any actinomycete, inthe genome of which pSAM2 vectors or its derivatives are capable ofintegrating. In particular, they may be used in fermentation processesinvolving strains of Streptomyces, of mycobacteria, of bacilli, and thelike. As an example, there may be mentioned the strains S.pristinaespiralis (ATCC 25486), S. antibioticus (DSM 40868), S.bikiniensis (ATCC 11062), S. parvulus (ATCC 12434), S. glauescens (ETH22794), S. actuosus (ATCC 25421), S. coelicolor (A3(2)), S. ambofaciens,S. lividans, S. griseofuscus, S. limosus, and the like (see alsoSmokvina et al., Applications of the integrated plasmid pSAM2, GIM90,Proceedings, Vol. 1, p. 403-407).

Vectors derived from pSAM2 containing the elements described above maybe constructed by a person skilled in the art on the basis of hisgeneral body of knowledge and the teachings of the present application(see also General techniques of molecular biology),

The sequences of the invention are most especially suitable for use inan industrial process for the production of antibiotics (spiramycin,streptogramins, β-lactams, and the like, the genes for which aredescribed in Applications EP 346,000 and PCT/FR93/00923, in particular.

The present invention will be described more fully by means of theexamples which follow, which are to be considered to be illustrative andnon-limiting.

LEGEND TO THE FIGURES

FIG. 1. Restriction map of plasmid pSAM2

FIG. 2. Restriction map of the vector pOS531

FIG. 3. Restriction map of the vector pOS532

FIG. 4. Restriction map of the vector pOS541

FIG. 5. Restriction map of the vector pOS544

FIG. 6. Restriction map of the vector pOS666. This vector is constructedfrom plasmid pPM927 (Smokvina et al., gene 94 (1990) 53) by insertion ofthe rmf gene (also designated pra) and the origin of replication ofpSAM2: tsr: thiostrepton resistance gene; stm/spc:streptomycin/spectinomycin resistance gene; oriR: E. coli origin ofreplication; oripSAM2: origin of replication of pSAM2; ter:transcription terminator.

FIG. 7. Activity of the vectors.

GENERAL TECHNIQUES OF MOLECULAR BIOLOGY

The methods traditionally used in molecular biology, such as preparativeextractions of plasmid DNA, centrifugation of plasmid DNA in a caesiumchloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, protein extractionswith phenol or phemnol/chloroform, ethanol or isopropanol precipitationof DNA in a saline medium, transformation in Escherichia coli, and thelike, are well known to a person skilled in the art and are amplydescribed in the literature Maniatis T. et al., "Molecular Cloning, aLaboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982; Ausubel F. M. et al. (eds), "Current Protocols in MolecularBiology", John Wiley & Sons, New York 1987!.

Plasmids of the pBR322 or pUC type and phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories).

For ligation, the DNA fragments may be separated according to their sizeby agarose or acrylamide gel electrophoresis, extracted with phenol orwith a phenol/chloroform mixture, precipitated with ethanol and thenincubated in the presence of phage T4 DNA ligase (Biolabs) according tothe supplier's recommendations.

The filling-in of 5' protruding ends may be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3' protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5'protruding ends is performed by a controlled treatment with S1 nuclease.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may beperformed according to the method developed by Taylor et al. NucleicAcids Res. 13 (1985) 8749-8764! using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by the so-called PCRPolymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis, K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350! technique may be performed using a "DNA thermal cycler"(Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences may be performed by the methoddeveloped by Sanger et al. Proc. Natl. Acad. Sci. USA, 74 (1977)5463-5467! using the kit distributed by Amersham.

EXAMPLES Example 1

Cloning and sequencing of the sequence SEQ ID No. 1

The free form of pSAM2 (B2) is not observed in S. lividans TK24 (Hopwoodet al., J. Gen. Microbiol. 129 (1983) 2257), only the integrated form isobserved. With pSAM2 (B3), the free form of the plasmid coexists withthe integrated form. Hybrid plasmids were from pSAM2 (B3) in whichdifferent regions were replaced by the equivalent regions originatingfrom pSAM2 (B2). This enables it to be shown that the mutation enablingpSAM2 (B3) to exist in free form was localized in a 2-kb KpnIrestriction fragment. The sequence of this KpnI fragment was determinedfor pSAM2 (B2) and pSAM2 (B3). A single nucleotide differs between thesetwo sequences: a G/C pair in pSAM2 (B2) is replaced by an A/T pair inpAM2 (B3). Sequence analysis showed that this mutation occurred upstreamof an open reading frame which extended further than the KpnI site. Thesequence of this open reading frame was determined; it is presented inthe sequence SEQ ID No. 1. This open reading frame, designated rmf (orpra), is located between the korSA and traSA genes. The mutation causesthe disappearance of a recognition site for the restriction enzyme ApaLI(recognition site: 5'GTGCAC 3'). This site is present in pSAM2 (B2) butabsent in pSAM2 (B3).

The sequence SEQ ID No. 1 also comprises 100 bp upstream of the codingregion, comprising a protion of the promoter (residues 1 to 101) and the5' non-coding but transcribed region (residues 102 to 154) carrying, inparticular, the ribosome binding site (RBS).

In the mutant pSAM2 (B4, for which the free form can be observed, theApaLI site is still present, indicating that the mutation is notlocalized at the same place as in pSAM2 (B3). The sequence of pSAM2 (B4)for the corresponding region showed that the sequences of pSAM2 (B2) andof pSAM2 (B4) differed by one nucleotide. This mutation was localized 8nucleotides upstream of the mutation detected in pSAM2 (B3). Thus, intwo independent cases, the presence of the free form of pSAM2 (B3) andpSAM2 (B4), which coexists with the integrated form, is due to a pointmutation upstream of a pSAM2 gene. These two independent mutations areboth located in a region shown by messenger RNA analysis experiments toconstitute the promoter of this gene.

Example 2 Construction of cassettes and vectors for the portion of thesequence SEQ ID No. 1

Several constructions were carried out using the following promoters andplasmids:

Modified ermE promoter (ermE*). The modified ermE promoter usedcorresponds to the promoter described by Bibb et al. cited above,possessing a deletion of 3 nucleotides (bases 252-254, FIG. 2 of Bibb etal. cited above). This modified promoter gives a strong and constitutiveexpression. It was isolated in the form of a 275-bp KpnI-BamHI fragment.

tipA promoter. This promoter (Murakami et al. cited above) is present inplasmid pPM927 (Smokvina et al., Gene 94 (1990) 53). This is inducedspecifically in the presence of thiostrepton.

Plasmids pIJ486 and pIJ487. These two plasmids have been described byWard et al. (Mol. Gen. Genet. 203 (1986) 468). These plasmids alone haveno influence on the status (free or integrated) of pSAM2.

2.1. Construction of the vector pOS531

The vector pOS531 was constructed by cloning the coding portion of thermf gene (residues 155 to 505 of the sequence SEQ ID No. 1) at theBamHI-HindIII sites of plasmid pIJ487. This vector hence carries thenaked rmf gene (without expression signal or 5' non-coding region). Amap of this vector is given in FIG. 2.

2.2. Construction of the vector pOS532

The vector pOS532 was constructed by cloning the 275-bp fragmentcarrying the modified ermE promoter at the EcoRI-BamHI sites of thevector pOS531. This vector hence carries the naked rmf gene under thecontrol of the modified ermE promoter (FIG. 3).

2.3. Construction of the vector pOS541

The vector pOS541 was constructed in 2 steps:

cloning of a fragment containing the coding portion of the rmf gene andthe 5' non-coding region (residues 102 to 505 of the sequence SEQ IDNo. 1) at the BamHI-HindIII sites of plasmid pIJ487,

addition of the 275-bp fragment carrying the modified ermE promoter atthe EcoRI-BamHI sites of the vector obtained above.

This vector hence carries the rmf gene provided with its 5' non-codingregion, under the control of the modified ermE promoter (FIG. 4).

2.4. Construction of the vector pOS544

The vector pOS544 carries an rmf gene deleted in its 3' portion(residues 102 to 276 of the sequence SEQ ID No. 1 are present). It wasconstructed by cloning this region in the form of a BamHI-BspHI fragment(BspHI end treated with the Klenow fragment of E. coli DNA polymerase I)into the BamHI-HindIII sites of plasmid pIJ487 (HindIII end treated withthe Klenow fragment of E. coli DNA polymerase I). The vector pOS544hence carries the 5' portion of the rmf gens (5' non-coding region +5'coding region), which portion lacks, however, its promoter. A map ofthis vector is given in FIG. 5.

Example 3 Appearance of replicative free forms of integrative vectorsderived from pSAM2due to the expression product of the rmf gene

This example shows that the overexpression of the gens SEQ ID No. 1 cancause the appearance in free form of an integrated copy of pSAM2.

3.1. Direct evidence for the role of replication activator played by rmfmay be obtained by causing, by expression of rmf, the appearance in freeform of an integrated copy of pSAM2(B2). The fact that pSAM2(B2)possesses an additional ApaLI site relative to pSAM2(B3) enables it tobe verified simply by observation of the ApaLI digestion profile thatthe free plasmid is indeed pSAM2(B2).

3.2. The activity of the sequence SEQ ID No. 1 was also demonstrated bytransformation of S. lividans carrying an integrated copy of pSAM2(B2)with the different vectors described above, followed by a search forfree forms. To this end, S. lividans containing plasmid pSAM2(B2) wastransformed by the protoplast technique with the different vectorsdescribed in Example 2. The plasmid DNA or total cellular DNA isextracted from a stationary-phase culture of the transformed clones. Theplasmid DNA is digested with the enzyme ApaLI and the digestionfragments separated by agarose gel electrophoresis. Observation of therestriction profile reveals whether or not free copies of pSAM2(B2) arepresent. These results are then confirmed by experiments involvinghybridization of the total DNA with a pSAM2probe.

Results obtained are presented in FIG. 7.

These results show that pOS541, which contains the modified ermEpromoter, the untranslated region upstream of rmf with the ribosomebinding site and the rmf coding frame, causes the appearance ofreplicative free forms of pSAM2(B2). As expected, no effect is observedwith a vector containing only the rmf coding portion (pOS531), or with avector containing modified ermE and the coding portion of rmf but noribosome binding region (pOS532). No effect is observed with a vectorcarrying a deletion in the 3' portion of rmf (pOS544).

The same type of effect is obtained when the cassette permittinginducible expression of rmf is localized on the plasmid for which it isdesired to cause the appearance of free forms (vector pOS666, FIG. 6).

These results collectively provide a clear demonstration that thesequence of the invention is capable of inducing, in cis or in trans,the appearance of free forms of vectors derived from pSAM2.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 812 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCAGCAGCCGACTGACGACCGCTCAACTCCTCACAGCCCGTCGCGAGTTCTCTGTCGCGG60                CGGGTTGACTCATGTATAGGAGTGGTGCACTCTTCTTCATGTCACTCATATACATGAGTG120               ACGGAGTCCAGCCTCTATAGAGGAGTGATCCGCTGTGCGTCAGATCCCCGTCGACACCTC180               CGCCGCAACCGTGATGGTCGCCAAGACTCCGGAGCCGAAGGTGAAGGACCGCCGGACCGG240               TGAGCTGGCCGTCGACGCCGAGACCGGTGCCAAGCTCATGACCGTGAACGTGATGTTCGC300               GGCCAACGACGAAGTCGAGATTCTGTCCGTGACCGTCCCGGAGACCGGTATCTCCGGTGA360               ACTGGCCATGGGTACCCCGGTCGCGCTGACGGGGCTCATCGCCCGGCCGTGGGAGAACGA420               GTTCAACGGCCAGAAGCGGCACGGCATCGCGTTCCGCGCGGTCGCGGTCACGTCGCTGAC480               CGCTGCGGGCTCGAAGGCTGCCTGATCATGACGTGGTTCATGGTCGCTGTGGTTGTGGTC540               GTCGCTGCTGCGGGTCTCCTGCGGTGGCGGCGCCCCGCCTGGTACTGGCTCACCTTCGGG600               GCCCTGGTCGCGACGGTGCGGGTCCTGGTCCGTACGCCTCGGTCATGGAAGCGTGCGGGC660               TGACGGTCCGCCCTCACGCTGGCGGCTGCTCTGGCCCGGATGGCGAATGCCGCGCCTGAG720               TCCCGGCCGCCGCGCATCTTGCGGTTACGTCCCACTCGTACCGGCCTGGTCTGCGGCTCA780               AGCTCCGGCCGGGACAGGATGCCTTCGACGTG812                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgGlnIleProValAspThrSerAlaAlaThrValMetValAla                              151015                                                                        LysThrProGluProLysValLysAspArgArgThrGlyGluLeuAla                              202530                                                                        ValAspAlaGluThrGlyAlaLysLeuMetThrValAsnValMetPhe                              354045                                                                        AlaAlaAsnAspGluValGluIleLeuSerValThrValProGluThr                              505560                                                                        GlyIleSerGlyGluLeuAlaMetGlyThrProValAlaLeuThrGly                              65707580                                                                      LeuIleAlaArgProTrpGluAsnGluPheAsnGlyGlnLysArgHis                              859095                                                                        GlyIleAlaPheArgAlaValAlaValThrSerLeuThrAlaAlaGly                              100105110                                                                     SerLysAlaAla                                                                  115                                                                           __________________________________________________________________________

We claim:
 1. An isolated nucleic acid comprising all or part of thesequence SEQ ID NO. 1 or a variant of the latter, which sequence orvariant of said sequence is capable of inducing the appearance ofreplicative free copies of pSAM2 or its derivatives and wherein saidsequence or variant of said sequence is under the control of a promoterfunctional in actinomycetes wherein the promoter is a heterologousconstitutive promoter or a heterologous regulated promoter.
 2. Anexpression cassette comprising an isolated nucleic acid comprising allor part of the sequence SEQ ID NO. 1 or a variant of the latter, whichsequence or variant of said sequence is capable of inducing theappearance of replicative free copies of pSAM2 or its derivatives andwherein said sequence or variant of said sequence is under the controlof a constitutive or regulated promoter functional in actinomycetes. 3.The expression cassette according to claim 2, wherein the constitutivepromoter is chosen from the promoter of the ermE gene or a variant ofthe latter, the p14 promoter of phage I19 of S. ghanaensis, or anyfragment containing a promoter region of a ribosomal operon of S.ambofaciens.
 4. The expression cassette according to claim 2, whereinthe regulated promoter is chosen from promoters induced specifically byan agent introduced into the culture medium.
 5. The expression cassetteaccording to claim 2, wherein the regulated promoter is chosen fromactinomycetes promoters which are specifically active in the late phasesof the proliferation cycle of actinomycetes.
 6. The expression cassetteas claimed in claim 4, wherein the promoter is thethiostrepton-inducible promoter tipA.
 7. The expression cassette asclaimed in claim 4, wherein the promoter is a thermoinducible promoter.8. The expression cassette as claimed in claim 7, wherein the promoteris from groEL genes.
 9. The expression cassette as claimed in claim 5,wherein the actinomycetes promoter is a promoter of genes of secondarymetabolism.
 10. The expression cassette as claimed in claim 5, whereinthe actinomycetes promoter is a promoter of genes for the production ofantibiotics.
 11. A method for inducing the appearance of free copies ofvectors derived from pSAM2, comprising the following steps:(a) isolatingat least part of the sequence SEQ ID NO:1 or a variant of the sequence;(b) inserting at least part of the sequence or a variant of the sequenceinto an expression cassette; (c) transfecting the expression cassetteinto a host strain under conditions permitting expression of thesequence.
 12. The method as claimed in claim 11, wherein the expressioncassette is inserted into a vector before transfection into a hoststrain.
 13. The method as claimed in claim 12, wherein the vector isderived from pSAM2.
 14. The method as claimed in claim 11, wherein thehost strain is actinomycete.
 15. The method as claimed in claim 14,wherein the actinomycete is selected from the group consisting ofStreptomyces, mycobacteria, and bacilli.
 16. The method of claim 15further comprising the step of producing an antibiotic.